Normal human hemoglobin (or "haemoglobin") is a protein having a molecular weight of approximately 68,000 Daltons. Hemoglobin comprises four globin chains, each with a heme group attached; two of the globin chains in normal hemoglobin are referred to as ".alpha.(alpha)-chains", while the other two non-.alpha. chains are selected from .beta.(beta), .gamma.(gamma) or .delta.(delta) chains. The resulting four chain molecule can be referred to as a "tetramer". Typically, the hemoglobin types are designated based upon these chains, i.e. .alpha..sub.2 .beta..sub.2 or .alpha..sub.2 .delta..sub.2 indicating two .alpha. chains, two .beta. chains and two .alpha. chains, two .delta. chains, respectively. The hemoglobins present in a normal adult are Hemoglobin A (.alpha..sub.2 .beta..sub.2), which comprises about 97% of the total hemoglobin, Hemoglobin A.sub.2 (.alpha..sub.2 .delta..sub.2), and Hemoglobin F(.alpha..sub.2 .delta..sub.2), which comprise the remaining 3%.
The orientation of these four chains is such that the hemoglobin moiety comprises a cleft, or "pocket", on its exterior. This cleft contains the site of oxygen uptake and release, which is the principal function of hemoglobin. It is the interaction of the four globin chains which allows for alteration in spatial relationships between portions of the molecule, and this facilitates the uptake or release of oxygen. Abnormalities in the structure of the hemoglobin molecule can result in an inability to, e.g., properly regulate the uptake and release of oxygen.
The .alpha. chains comprise 141 amino acids and the non-.alpha. chains comprise 146 amino acids. The exact type, number and precise sequence of these amino acids is characteristic for each type of globin chain; any alteration in the sequences gives rise to an abnormal globin chain, resulting in the production of abnormal hemoglobin. Abnormal hemoglobins are part of a group of inherited disorders collectively referred to as the "hemoglobinopathies."
Hemoglobinopathies are the result of defective synthesis of the globin chains which form the hemoglobin molecule. Such a defect can occur in at least two ways: (1) synthesis of a structurally abnormal globin chain at the genetic level, and (2) decrease production of a structurally normal globin chain whereby the .alpha. and non-.alpha. chains are synthesized in unequal quantities, and the resultant imbalanced chain production causes an inadequate production of the normal hemoglobin, which forms an unstable tetramer. Conditions which arise from such imbalanced chain production are referred to as the "thalassemia syndromes."
With reference to structural defects, approximately 400 structural abnormalities of the globin chain have been described. The vast majority of these provide neither clinical nor hematologic manifestations. These hemoglobin structural variants are due principally to point mutations, insertions or deletions of the nucleotides encoding the globin genes, or deletions or fusion of these genes. Hemoglobin S (HbS) is by far the most important abnormal hemoglobin variant in terms of clinical significance. Individuals having two genes for HbS ("homozygous" for HbS) have sickle-cell anemia and will be at risk of severe life threatening crises. The phrase "sickle-cell" is derived from the observed sickle-shaped red blood cells characteristic of HbS. Individuals with sickle cell anemia can experience painful sickling crises when the sickle cells cause blockage of the arteries, resulting in vasa-occlusion and tissue infarctions.
With reference to the second type of hemoglobinopathy, the thalassemia syndromes, a reduction as .alpha.-chain synthesis results in a condition referred to an ".alpha.-thalassemia". Alpha-thalassemia is considered to be the most common genetic disorder in humans. Clinical expressions of .alpha.-thalassemia range from none to very severe; these expressions can be determined by analysis of the hemoglobins. I.e., individuals having three deleted .alpha.-genes typically evidence an increase in Hemoglobin F and the appearance of Hemoglobin H. Beta-thalassemias have similar varying degrees of clinical expressions and these can also be determined by analysis of the hemoglobins, i.e. for certain .beta.-thalassemias, no Hemoglobin A is present, while for .beta..delta.-thalassemia, Hemoglobin F is characteristically increased to 5-20% of total hemoglobin.
Many of the hemoglobinopathies are race specific. For example, about 30% of African-Americans and persons of southeast Asia origin have a gene for .alpha.-thalassemia. Beta-thalassemia occurs frequently in Mediterranean and Asian individuals.
Screening for the presence of abnormal hemoglobins is typically conducted with the intent of detecting both clinically significant and clinically silent abnormal hemoglobins. For example, it is essential to screen any person of non-Northern European origin who is undergoing an anaesthetic procedure for the presence of HbS. This is because the presence of HbS indicates a possible inability to properly regulate the uptake of oxygen such that appropriate anaesthetic procedures are utilized. Such screening is also useful in genetic counseling. Individuals who, for example, are "heterozygous" for the HbS gene (one gene for HbS; sickle cell carrier) have sickle cell traits and can experience no clinical manifestations. However, when two such individuals pro-create, their offspring have a 50% chance of inheriting both HbS genes, i.e. a 50% chance of being heterozygous for HbS and having sickle cell anemia.
There are currently two principal approaches utilized for the separation and detection of hemoglobin variants: a) slab-gel electrophoresis and b) isoelectric focusing. Both protocols are based upon the ability of hemoglobin variants to be separated from each other due to the difference in electrophoretic mobilities of such variants. The amino acid differences in the composition of hemoglobin variants are responsible for the differences in electrical charge, and this produces the difference in electrophoretic mobility. Under the influence of a charged field, all of the variants will move toward a designated charge opposite to the charge of the variants; those having a lower electrophoretic mobility will move slower than, and hence be separated from, those having a (relative) higher electrophoretic mobility.
Both of the foregoing protocols are based on the use of some type of gel material as the separating medium. In slab-gel electrophoresis, the sample is placed upon a suitable electrophoretic medium (e.g., paper, cellulose acetate membranes, agarose, etc.). The medium is electrophoresed at a suitable voltage to cause separation of the variants; the electrophorsized gels are then fixed and stained. This procedure is somewhat complex, requires skill, and can require up to about one hour to obtain clinically useful results. Examples of well recognized and widely accepted types of slab gel electrophoretic protocols are the PARAGON.RTM. slab gel electrophoretic system and APPRAISE.RTM. densitometer electrophoresis systems (available from Beckman Instruments, Inc., Fullerton, Calif. USA). For slab gel electrophoretic separations of hemoglobin variants, Hemoglobin A, Hemoglobin F, Hemoglobin S and Hemoglobin C have been separated using a basic buffer (e.g., a 50mM barbital buffer, pH 8.6). However, in order to achieve unequivocal separation of Hemoglobin S from co-migrating variants (e.g., Hemoglobins D and G), an acidic buffer must be used (e.g., 70mM maleate, pH 6.0).
Isoelectric focusing allows for the simultaneous separation of Hemoglobins A, F, S and C. See, e.g., Zhu, M. et al. "Optimizing Separation Parameters in Capillary Isoelectric Focusing." J. Chrom. 559:479-488 (1991), which is incorporated herein by reference. However, isoelectric focusing is not a widely accepted methodology for such separations. This technique requires the use of a stable pH gradient such that the hemoglobin variants migrate via electrophoretic techniques to the zone where the pH is equal to the isoelectric point of the variant; in such a zone, the effective charge of the variant becomes zero, and migration ceases. Isoelectric focusing of hemoglobin variants has been applied using capillary electrophoresis techniques whereby a polyacrylamide coated microcapillary column comprising a buffer solution including carrier ampholytes (compounds which are both conductive and provide the necessary pH gradient) is used as the separation medium. Coating of the microcapillary column is essential for isoelectric focusing techniques. After the hemoglobin variants are separated in the various zones, an electrophoretic current is applied, and the separated variants are sequentially detected as they move through the capillary column past a detection system.
While capillary electrophoretic isoelectric focusing poses unique problems, capillary zone electrophoretic techniques are of interest. Capillary zone electrophoresis ("CZE") is a technique which permits rapid and efficient separations of charged substances. Separation of the constituents of clinical samples (i.e. whole blood, plasma, serum, urine, cerebrospinal fluid) can be accomplished in less than 20 minutes, typically in less than 10 minutes. In general, CZE involves introduction of a sample into a capillary tube, i.e. a tube having an internal diameter of from about 2 to about 2000 microns (".mu.m"), and the application of an electric field to the tube. The electric potential of the field both pulls the sample through the tube and separates it into its constituent parts. I.e., each of the sample constituents has its own individual electrophoretic mobility; those having greater mobility travel through the capillary tube faster than those with slower mobility. As a result, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones.
CZE can be generally separated into two categories based upon the contents of the capillary columns. In "gel" CZE, the capillary tube is filled with a suitable gel e.g., polyacrylamide gel. Separation of the constituents in the sample is predicated in part by the size and charge of the constituents travelling through the gel matrix. Gel CZE has several disadvantages, notably, the unpredictability of the gel material. I.e., such gels eventually "breakdown" or can only be used for limited analytical runs. Such unpredictability is unacceptable in any setting where numerous analytical runs are conducted.
In "open" CZE, the capillary tube is filled with an electrically conductive buffer solution. Upon ionization of the capillary, the negatively charged capillary wall will attract a layer of positive ions from the buffer. As these ions flow towards the cathode, under the influence of the electrical potential, the bulk solution (i.e., the buffer solution and the sample being analyzed), must also flow in this direction to maintain electroneutrality. This electroendosmatic flow provides a fixed velocity component which drives both neutral species and ionic species, regardless of charge, towards the cathode. The buffer in open CZE is as stable against conduction and diffusion as the gels utilized in gel CZE. Accordingly, separations can be obtained in open CZE quite similar to those obtained in gel-based electrophoresis.
Fused silica is principally utilized as the material for the capillary tube because it can withstand the relatively high voltage used in CZE, and because the inner walls ionize to create the negative charge which causes the desired electroendosmatic flow. However, alumina, beryllium, Teflon.RTM.-coated materials, glass, and combinations of these (with or without fused silica) can also be utilized. The capillary column is typically capable of withstanding a wide range of applied electrophoretic fields of between about 10 volts per centimeter ("v/cm") up to about 1000v/cm. The capillary column may be coated on the outside (using, e.g., a polyamide material) for ease of handling. The inner wall of the capillary may be untreated or coated with a material capable of, inter alia, reducing adsorption to the inner wall during electroendosmatic flow of the bulk solution. However, it is typically preferred that the inner wall be uncoated because typical coatings have a tendency to breakdown in an unpredictable manner.
Open CZE has many desirable qualities for clinical sample analysis: because the analysis does not involve a gel-filled column, the inherent limitations on the number of analytical runs that can be conducted with any particular gel-filled column are avoided; when the capillary column is untreated, the aura of unpredictability which can be associated with coated columns is avoided; the sample size is small (usually on the order of 5 to 200 .mu.l of diluted sample); sample analysis time is fast, i.e. less than about 20 minutes; and the protocol lends itself to automation, thus decreasing the labor skills necessary for efficient and effective sample analysis. Heretofore, the unequivocal separation of hemoglobin variants, and in particular hemoglobin variants A, F, S and C, has not been demonstrated using open CZE protocols.
Given to benefits associated with open CZE, and the necessity for screening for hemoglobin variants, it would be advantageous to have an open tube CZE protocol for analyzing hemoglobin variants.